118
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: not found

      A highly efficient recombineering-based method for generating conditional knockout mutations.

      1 , ,
      Genome research
      Cold Spring Harbor Laboratory

      Read this article at

      ScienceOpenPublisherPMC
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Phage-based Escherichia coli homologous recombination systems have recently been developed that now make it possible to subclone or modify DNA cloned into plasmids, BACs, or PACs without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombineering, has many different uses for functional genomic studies. Here we describe a new recombineering-based method for generating conditional mouse knockout (cko) mutations. This method uses homologous recombination mediated by the lambda phage Red proteins, to subclone DNA from BACs into high-copy plasmids by gap repair, and together with Cre or Flpe recombinases, to introduce loxP or FRT sites into the subcloned DNA. Unlike other methods that use short 45-55-bp regions of homology for recombineering, our method uses much longer regions of homology. We also make use of several new E. coli strains, in which the proteins required for recombination are expressed from a defective temperature-sensitive lambda prophage, and the Cre or Flpe recombinases from an arabinose-inducible promoter. We also describe two new Neo selection cassettes that work well in both E. coli and mouse ES cells. Our method is fast, efficient, and reliable and makes it possible to generate cko-targeting vectors in less than 2 wk. This method should also facilitate the generation of knock-in mutations and transgene constructs, as well as expedite the analysis of regulatory elements and functional domains in or near genes.

          Related collections

          Author and article information

          Journal
          Genome Res
          Genome research
          Cold Spring Harbor Laboratory
          1088-9051
          1088-9051
          Mar 2003
          : 13
          : 3
          Affiliations
          [1 ] Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA.
          Article
          10.1101/gr.749203
          430283
          12618378
          bc00fdbf-85f8-4936-9434-fd70466252b2
          History

          Comments

          Comment on this article